Giardia lamblia is the most common disease-causing parasite in the United States responsible for an estimated 3 million cases a year. Besides causing problems in day care centers, travelers, backpackers, and homosexuals, it not uncommonly contaminates water supplies and has been responsible for massive epidemics. The organism lives in the small intestines and causes diarrhea, abdominal pain, nausea and vomiting. Symptoms are commonly intermittent and long lasting. An environmentally resistant cyst form is passed in the feces, and, because large numbers of cysts are excreted and only a few can initiate infection, infections are common. Previously a system for stably placing DNA into Giardia was improved (stable transfection) so that enough protein could be expressed to make it easily detected in the parasite. This allows the expression of any protein in the organism and experiments to understand how proteins are made transported and secreted. A number of studies were performed using this new system.We were able to show that Giardia employs the same signals that higher mammalian cells use to import proteins into the nucleus.In higher mammalian organisms, proteins are made from RNA which itself is made from DNA. The rules that govern how RNA is made and controlled are not well defined in Giardia but are likely to differ compared to more developed organisms because a relatively small amount of DNA that precedes the coding region of the actual protein is all that appears to be necessary to control RNA production (called transcription). Analysis of the alpha-2 tubulin promoter region was analyzed and showed that the most important initiator is where the RNA begins to be made. The exact sequence did not appear to be as important as in other organisms. Other sequences upstream of the initiator seemed to control the degree of transcription. In another series of experiments similar studies were performed using the glutamate dehydrogenase gene of Giardia. Here, 3 important regions were found within 44 bp of the coding region and a protein that bound to two of these regions was identified. Both the proteins studied above are made all the time by Giardia. However, other proteins are produced only during certain periods such as when Giardia encysts in preparation for survival outside the host. Using a previously defined cyst wall protein (cwp2), we showed that the regulatory sequences are within 100 bp of the coding region and that proteins controlling this bind close to the start of the coding region. Giardia has the ability to change its surface relatively frequently, a process called antigenic variation. Usually only one surface antigen or VSP can be seen on its surface. How only one VSP among many is expressed on the surface of the parasite, how VSPs are made and transported to the surface, and what is their biologic role are of major interest. Using the transcription system described above we have begun to explore the molecular mechanisms involved in antigenic variation. Antigenic variation occurs when only a limited region of the upstream region of VSP gene are present. Mutational studies designed to understand what portions of the VSP are important were continued.